Smad4 restricts injury-provoked biliary proliferation and carcinogenesis

ABSTRACT Cholangiocarcinoma (CCA) is a deadly and heterogeneous type of cancer characterized by a spectrum of epidemiologic associations as well as genetic and epigenetic alterations. We seek to understand how these features inter-relate in the earliest phase of cancer development and through the course of disease progression. For this, we studied murine models of liver injury integrating the most commonly occurring gene mutations of CCA – including Kras, Tp53, Arid1a and Smad4 – as well as murine hepatobiliary cancer models and derived primary cell lines based on these mutations. Among commonly mutated genes in CCA, we found that Smad4 functions uniquely to restrict reactive cholangiocyte expansion to liver injury through restraint of the proliferative response. Inactivation of Smad4 accelerates carcinogenesis, provoking pre-neoplastic biliary lesions and CCA development in an injury setting. Expression analyses of Smad4-perturbed reactive cholangiocytes and CCA lines demonstrated shared enriched pathways, including cell-cycle regulation, MYC signaling and oxidative phosphorylation, suggesting that Smad4 may act via these mechanisms to regulate cholangiocyte proliferation and progression to CCA. Overall, we showed that TGFβ/SMAD4 signaling serves as a critical barrier restraining cholangiocyte expansion and malignant transformation in states of biliary injury.


Fig. S2 .
Fig. S2.A) Quantification of inflammatory leukocytes in control and injured (DOC diet) livers of Ctl and Smad4 del mice showing no significant difference .Livers from mice on control diet (n=3 mice per cohort) and DOC diet (n=6 mice per cohort) were digested and analyzed for cells staining positive for the immune marker CD45.Representative FAGS plots are shown on the right.(B) Quantification of inflammatory monocytes/macrophages and granulocytes in control and injured (DOC diet) livers of Ctl and Smad4 del mice showing no significant difference.Livers from mice on control diet (n=3 mice per cohort) and DOC diet (n=6 mice per cohort) were digested and analyzed for cells staining positive for the following immune markers CD45+/CD11b+/Ly6C-high/Ly6G-low for monocytes/macrophages and CD45+/CD11 b+/Ly6C-high/Ly6G-high for granulocytes.Representative Ly6C/Ly6G plots are shown on the right.(C) Quantification of area staining positive for the myofibroblast marker aSMA compared to total liver revealing no significant difference .Random 40x fields were used for quantification , n=3 mice per cohort with 3 fields per mouse.Representative images are shown on the right (magnified 200x).(D) Quantification of collagen area staining positive for Sirius red showing no significant difference, n=3 mice per cohort .Representative images are shown on the right (magnified 100x).(E) Quantification of cells staining positive for apoptosis cleaved-caspase 3 (CC3) revealing no significant difference .Random 200x images were used for quantification, n=2-3 mice per cohort with 5 fields per mouse.Representative images are shown on the right (magnified 200x).ns, not significant ,*p<0 .05,Bar=25 µm in C and E, Bar=50 µm in D.

Fig. S3 .
Fig. S3.FACS-isolation enriches for reactive cholangiocytes.mRNA expression levels assessed by qPCR of hepatocyte and biliary markers in samples taken before the sort and after the sort, demonstrating enrichment of the biliary compartment.

Fig. S4 .
Fig. S4.(A) Macroscopic images of AKP and AKPS livers with representative histology images of control AKP CCA and HCC (magnified 100x with selected boxed areas shown enlarged on the right).(B) Representative images of positive CK7 staining and ARG1 staining in murine CCA and HCC, respectively (magnified 100x).(C) Macroscopic images of AP and APS with representative histology images of control AP livers with normal histology (magnified 100x with selected boxed area shown enlarged on the right).(D) Quantification of tumors and hamartomas in AP and APS mice.*p<0.01,Bar=50 µm.

Fig. S5 .
Fig. S5.(A) mRNA expression levels assessed by qPCR to characterize the differentiation state of AKPS cell lines in vitro.Included are normal liver to provide a representative hepatocellular signature and AKP cell lines derived from CCA (AKP iCCA #1 and #2) to represent a biliary signature.AKPS cell lines #1-5 show expression of biliary markers.Expression was normalized to Rhoa.(B) Gene set enrichment analysis (GSEA) of genes differentially expressed in Smad4-restored CCA cell lines.Enrichment plots from the Hallmarks Gene Set Collection are shown for the top five gene sets most negatively correlated (top row) in genes downregulated in Smad4-restored CCA cell lines or most positively correlated (bottom row) in genes upregulated in Smad4-restored CCA cell lines.(C) mRNA expression levels assessed by qPCR of MYC target genes and Ox-Phos related genes derived from the leading edge analysis of reactive cholangiocyte GSEA.Expression was normalized to Rhoa.Ox-Phos, oxidative phosphorylation; EMT, epithelial mesenchymal transition; NES, normalized enrichment score.

Fig. S6 .
Fig. S6.Hierarchical clustering of DNAme changes in Smad4 restored CCA cell lines.Pearson correlation coefficients were determined for pairwise comparisons of all genome-wide DNAme (RRBS) datasets.Hierarchical clustering was then applied to group samples to distinguish similarity in datasets.A colored heatmap scale is shown to indicate Pearson R values.